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Drought is one of the major abiotic stresses limiting crop productivity in arid and semi-arid regions and influences many aspects of plant development. Groundnut (Arachis hypogaea L.) is an important oil yielding crop and considered as relatively drought tolerant. In this study, two groundnut cultivars were first tested for their drought tolerance based on physiological marker attributes such as relative water content, total chlorophyll content, cell membrane stability and free proline content and identified cultivar K-134 as a drought tolerant and cultivar JL-24 as drought susceptible. To gain a better understanding of the drought stress responses at molecular level, we carried out a genomic analysis of stress-responsive genes/transcripts in drought-tolerant cultivar K- 134. As a first step toward characterization of stress-responsive genes, construction and analysis of subtracted cDNA library from droughttolerant cultivar (K-134) is reported here. Using this strategy a total of 200 ESTs were isolated, sequenced, of which 120 high-quality ESTs were obtained and clustered. Further, our analysis revealed that 31% sequences were unique and no homology to known proteins in the database. This observation has great relevance since groundnut is a stress- adapted legume crop. Further, to validate the identified differentially expressed genes, expression profiles of selected clones were analyzed using dot blot (reverse northern), northern blot analysis. We showed that these clones are differentially expressed under different abiotic stress conditions. The implications of the analyzed genes in abiotic stress tolerance were discussed.
The NAC gene family encodes plant-specific transcription factors which play diverse roles in abiotic stress responses of plants. NAC TFs reported to be involved in the regulatory pathway of multiple abiotic stresses. In the present study, a salt stress-inducible NAC gene, named MuNAC4 (Macrotyloma uniflorum NAC4) from horsegram was isolated, cloned, characterized and studied its expression. Real-time PCR expression analysis showed up-regulation of MuNAC4 in horsegram across salinity, cold, drought and dehydration stress conditions. However, salt stress resulted a sixfold increase in MuNAC4 transcript levels. The involvement of MuNAC4 in abiotic stress tolerance was investigated by cloning MuNAC4 gene in expression vector pET28a and transformation in Escherichia coli BL21 (DH3). The apparent molecular weight of the recombinant protein was found to be 38.3 kDa as evident from SDS-PAGE analysis. The functional role of MuNAC4 in Escherichia coli confers the tolerance against salt (6 % NaCl), heavy metal (100 mM CuSO4) and water stresses (6 % PEG). The E. coli cells transformed with pET28a ? MuNAC4 was showed better survival ratio and higher growth rates under NaCl stress than other abiotic stress conditions when compared to control cells (BL21/ pET28a). This provides the experimental evidence that MuNAC4 protein enhance salt stress tolerance of E. coli cells suggesting their suitability as candidates for genetic manipulations for enhanced crop tolerance to salt stress and other abiotic stresses.
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